DNA vaccination represents a unique strategy to overcome the limitations of immunization with conventional vaccines which is restricted by the high variability of influenza viruses. We evaluated the protective efficacy of a plasmid DNA (pDNA), encoding an evolutionarily conserved epitope of viral matrix protein, against the influenza A virus infection. It was found that the mice immunized via the intra-muscular route purely elicited cell mediated immune response to the pDNA, with enhanced level of Th1 cytokines viz. IL-12 and IFNγ production in the stimulated splenocyte supernatant. The cytotoxic T lymphocytes in the spleen of immunized mice significantly lysed the virus-infected MDCK cells. A significant decrease in virus replication was also observed in the lungs of immunized mice and 83% of the mice were protected against the lethal challenge of influenza A viruses. These findings suggest that the plasmid DNA expressing a single matrix epitope may serve as a promising vaccine candidate to provide effective immunity in the susceptible (mouse) population.